Phillip A. Sharp

Bio: Phillip A. Sharp is an academic researcher from Massachusetts Institute of Technology. The author has contributed to research in topics: RNA & RNA splicing. The author has an hindex of 172, co-authored 614 publications receiving 117126 citations. Previous affiliations of Phillip A. Sharp include McGovern Institute for Brain Research & Medical Research Council.

Regulation of adenovirus mRNA synthesis

Abstract: The lytic cycle of adenovirus is a tightly regulated sequence of stages. When this regulation is studied at the level of mRNA production, the most significant step in controlling gene expression is initiation of transcription. Thus in preceding from one stage of expression to another, viral factors seem to turn on transcription of new sets of genes. At the moment, it is thought that viral mRNA synthesis involves initiation of transcription at ten different promoter sites. It is likely that in some manner the frequency of an initiation of transcription at nine of these sites is affected by one or more viral gene products. With the recent development of soluble in vitro transcription systems that respond to exogenously added DNA, it should be possible to begin to study regulation of gene expression at this stage of transcription. At present, these systems yield the paradoxical observation that extracts prepared from uninfected human cells more efficiently recognize the late promoter as compared to the early promoter of adenovirus. As more is learned about regulation of synthesis of viral mRNAs, examples will surely be found where RNA processing and RNA turnover play a critical role in determining the level of mRNAs. Such cases are more likely to appear in the balancing of synthesis of different mRNAs derived from one transcriptional unit. Few experiments have been directed to this possibility and the study of adenovirus molecular biology is only now entering the age of maturity where these experiments are feasible.

Sequential transcription-translation of simian virus 40 by using mammalian cell extracts.

Abstract: Ribonucleic acids (RNAs) transcribed in vitro by using the whole-cell extract system of Manley et al. (Proc. Natl. Acad. Sci. U.S.A. 77:3855-3859, 1980) were tested for their efficiency and fidelity in directing protein synthesis in reticulocyte lysates. Simian virus 40 deoxyribonucleic acid (DNA), cleaved by various restriction endonucleases, was used as the template. Successful translation of the small tumor antigen t, as well as the capsid proteins VP1, VP2, and VP3, was detected by immunoprecipitation analysis. Although no synthesis of large T antigen was detected, use of this technology allows detection of large T synthesis resulting from the correct splicing of as little as 0.2% of the in vitro RNA transcripts, making it ideal for use as an in vitro splicing assay. Transcripts synthesized in vitro were used as messages at least as efficiently as were viral messenger RNA's (mRNA's) synthesized in vivo; and in the case of small t, there was more efficient translation of small t mRNA synthesized in vitro than of small t mRNA synthesized in vivo. The transcripts that served as mRNA's for the various polypeptides were identified by using the following two criteria. (i) The sensitivity of synthesis of a given protein to digestion of the template DNA with restriction enzymes allowed the localization of the promoter and coding regions. (ii) Translation of size-fractionated RNA allowed confirmation of the transcript-mRNA assignments. With these techniques we found that VP2, VP3 and, in some cases, VP1 synthesis resulted from the initiation of translation at internal AUG codons. In fact, families of polypeptides were produced by initiation of translation at AUG codons within sequences coding for VP1 and T, presumably as a result of transcription initiation events that generated 5' ends immediately upstream from these AUGs. Application of this technology for the identification of coding regions within cloned DNA fragments is discussed.

Light Scattering and Hydrodynamic Properties of Polymer Chains with Excluded Volume Effects

Abstract: This paper considers the implications of the Domb–Gillis–Wilmers distribution function PN(R) = CNRδexp−(R / σ)δ for end‐to‐end distances R in polymer chains of N segments with excluded volume effects. CN is a normalizing constant; e is related to the standard deviation of R; and, according to Fisher, δ = 2 / (1 − e), where e is defined by 〈R2〉 = b2N1+e for polymers with step length b. We have calculated the angular dependence of light scattering, the translational frictional coefficient and intrinsic viscosity, and the dimensional statistics of cyclic chains. Comparison has been made with a previously used distribution function which took excluded volume effects into account in a less well‐founded way. The present distribution function gives properties which differ measurably from those obtained with the previous one, reflecting the greater average expansion of the chain implied by the distribution proposed by Domb et al.

Enhancer features that drive formation of transcriptional condensates

Abstract: Summary Enhancers, DNA elements that regulate gene expression, contain transcription factor (TF) binding sites. TFs bind short sequence motifs that are present throughout the genome at much higher frequency than active enhancers, and so the features that define active enhancers are not well understood. We show that DNA elements with TF binding site valency, density, and binding affinity above sharply defined thresholds can recruit TFs and coactivators in condensates by the cooperative process of phase separation. We demonstrate that weak cooperative interactions between IDRs of TFs and coactivators in combination with specific TF-DNA interactions are required for forming such transcriptional condensates. IDR-IDR interactions are relatively non-specific with the same molecular interactions shared by many TFs and coactivators, and phase separation is a universal cooperative mechanism. Therefore, whether a genomic locus is an enhancer that can assemble a transcriptional condensate is determined predominantly by its cognate TFs’ binding site valency and density.

Initial sequencing and analysis of the human genome.

Abstract: The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Abstract: 抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

A rapid alkaline extraction procedure for screening recombinant plasmid DNA

Abstract: A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.